Notch is an important regulator of gene transcription involved in cellular differentiation, lateral inhibition and tissue homeostasis. In Notch signaling the Notch precursor protein is activated in the trans-Golgi-network by proteolytic cleavage. This cleavage generates two fragments that comprise the active Notch-receptor, which is then translocated to the plasma membrane (PM). Upon interaction with its ligand from the Delta/Serrate/Jagged family it is subsequently processed by ADAM 17 and finally by gamma-secretase to generate the Notch intracellular domain (NICD). NICD is translocated to the nucleus, where it activates a transcription factor of the CSL family (CBF1 (RBP-J) in vertebrates, Su(H) in Drosophila, Lag-1 in C. elegans). NICD is composed of several Ankyrin repeats and an N-terminal RAM domain, which is critical for the activation of CSL (for review see1). Furthermore, one characteristic and important hallmark of the Notch signaling pathway is the independence of second messengers. Thus, the amount of NICD inside the nucleus is proportional to the amount of processed Notch receptor.
The final cleavage step that releases NICD from the PM is mediated by gamma-secretase. gamma-secretase is a heteromultimeric integral membrane protein with protease activity2. Interacting and potentially regulating proteins were shown3-6 but a commonly accepted view of the regulation of gamma-secretase is lacking. Notch signaling can be fine-tuned at several steps, ranging from glycosylation at the ectodomain, interaction with inhibitors like Numb, ubiquitinylation and phosphorylation of NICD to slight variations at the gamma-secretase cleavage site (for review see1). Fine-tuning can regulate trafficking of the receptor, ligand binding-affinity, endocytosis rate of the receptor and stability of NICD1,7. Despite the wealth of information about Notch signaling, its trafficking and regulation of signaling is not fully understood.
Therapeutic interventions of aberrant Notch signaling, for example in cases of T cell-lineage acute lymphoblastic leukemia (T-ALL) at present are aimed/tested at the level of ligand-binding and gamma-secretase cleavage, but novel strategies would be highly desired8,9.
Natural compounds like Brefeldin A (BFA), Shiga- or Cholera-toxin turned out to be invaluable tools to dissect molecular details of membrane trafficking at various steps in the exo- or endocytic pathway10.
Similarly, gamma-secretase related research could not have come this far without the identification of gamma-secretase inhibitors (GSI) such as DAPT or L685,458. These and other GSIs turned out to be not only invaluable to inhibit gamma-secretase but also to purify it, elucidate structure-function relations within the gamma-secretase complex and identify substrate-docking sites (13).
High content screening (HCS) describes the process of automated image acquisition of phenotypes of cells or organisms and the subsequent automated analysis of these phenotypes by image analysis algorithms without or only little user intervention allowing for high throughput applications such as genome-wide RNAi or large chemical compound libraries (for review see 14).
Despite various innovative screening strategies having been applied, there is a need in the field to find further inhibitors of the notch signaling pathway for application in medicine.
GSIs have been applied in the treatment of cancer, based on the finding that Notch signaling is misregulated in various cancers. The Notch signalling pathway is an important component in the molecular mechanisms that regulate cell fate during development, in addition to cancer formation. Aberrant activation of the Notch pathway contributes to tumour formation. The important role of Notch in human cancer has been highlighted by the presence of activating mutations and amplification of Notch genes in human cancer and by the demonstration that genes in the Notch signalling pathway could be potential therapeutic targets. One of the major therapeutic targets in the Notch pathway are the Notch receptors, in which γ-secretase inhibitors prevent the generation of the oncogenic (intracellular) domain of Notch molecules and suppress Notch activity. Further notch inhibitors are therefore sought after as potential cancer therapeutics.
Despite the promising effects of gamma-secretase inhibitors, known inhibitors are plagued by significant side effects during and after treatment of patients. Major side effects relate to gastrointestinal toxicity, such as nausea, diarrhea, vomiting, weight loss and/or loss of appetite.
The present invention therefore provides novel compounds not previously identified as Notch inhibitors. Furthermore, the compounds of the present invention show an anti-secretion function and play a role in membrane trafficking, inhibiting secretion at a pre-ER exit step. The effect of administration of the compounds of the invention in pre-clinical assays shows a notch phenotype in addition to disruption of secretion at an early stage. This effect enables the compounds as disclosed herein to be administered in the treatment of diseases associated with secretion or secretion pathways.
One example of such a condition is senescence-related ageing, whereby senescent cells lose their proliferative capacity once senescence occurs and show an enhanced secretion of pro-inflammatory cytokines, which is also known as the “senescence associated secretory phenotype (SASP):
Accumulation of nuclear DNA damage represents one of the molecular causes of aging. Accordingly, there is an age-dependent accumulation of DNA damage in numerous human tissues and genetic diseases that lead to premature aging are often caused by mutations in genes involved in DNA damage repair; and animal models provided a proof of concept that DNA damage accumulation leads to premature aging. Cells respond to DNA damage by activating checkpoints that prevent the contribution of damaged cells to tissue homeostasis by induction of cell death (apoptosis), cell cycle arrest (senescence) or self-digestion of the damaged cells (autophagy).
Senescent cells lose their proliferative capacity and show an enhanced secretion of pro-inflammatory cytokines, which is also known as the “senescence associated secretory phenotype (SASP)”. It has been shown that SASP affects neighbouring, non-senescent cells and this may impact on tissue aging and cancer formation. Methods or means for the inhibition of SASP could delay tissue dysfunction and extend healthy lifespan.
Little is known about the SASP-underlying changes in structure and composition of the secretory pathway. Senescence is associated with morphological changes in the secretory pathway like dispersal of the Golgi, increase in lysosomal volume, increase in lysosomal lipofuscin aggregation and increased expression of the lysosomal enzyme β-galactosidase. In light of the secretion phenotype associated with SASP, inhibitors of secretion represent a promising means for treating illness associated with SASP. At the present time no effective therapeutic approaches for the treatment of SASP-associated disease are known, and appropriate secretion inhibitors for the treatment of such disorders are required.
The compounds of the present invention therefore relate to FLI-06 and structurally similar derivatives that exhibit the properties desired in compounds for treatment of secretion-dependent disease, such as cancer or ageing.
Structurally similar compounds have been previously disclosed in relation to different biological effects or for the treatment of different medical conditions. WO 2009/102864 discloses structurally similar compounds to those of the present invention as antagonists of the hedgehog signaling pathway. However, the compounds disclosed therein are not mentioned in relation to secretion or notch signaling inhibition and additionally show different structural features, for example the ester position of R2 of formula I of the present invention is distinct from those compounds of WO 2009/102864, which exhibit an alkoxy methoxyethyl group at the corresponding position.
WO 2008/103470 discloses structurally similar compounds to those of the present invention as RAS-specific anti-cancer agents. However, the compounds disclosed therein are not mentioned in relation to secretion or notch signaling inhibition and additionally show different structural features, for example the ester position of R2 of formula I of the present invention is distinct from those compounds of WO 2008/103470, which exhibit an alkoxy group at the corresponding position. Furthermore, the compounds of WO 2008/103470 exhibit a phenyl substituent in place of R6. Additionally the compounds disclosed therein exhibit a different central ring structure compared to the compounds of formula I of the present invention.
WO 2011/050353 discloses structurally similar compounds to those of the present invention for the treatment of androgen-receptor-positive cancer cells. However, the compounds disclosed therein are not mentioned in relation to secretion or notch signaling inhibition and additionally show different structural features, for example the ester position of R2 of formula I of the present invention is distinct from those compounds of WO 2011/050353, which exhibit a phenylethyl group. It is noteworthy that none of the compounds disclosed in the art that exhibit structural similarity to the presently claimed compounds and are intended for cancer treatment exhibit a substitution of the hexahydroquinoline ring at position 7 with two methyl groups.
WO 2008/070875 also discloses structurally similar compounds to those of the present invention. The compounds of WO 2008/070875 are only disclosed in relation to the treatment of Alzheimer's disease and are not mentioned in relation to secretion or notch signaling inhibition.